Biological attributes required for epidermal regeneration: Evaluation of the next-generation autologous cell harvesting device.

Early wound intervention and closure is critical for reducing infection and improving aesthetic and functional outcomes for patients with acute burn wounds and nonthermal full-thickness skin defects. Treatment of partial-thickness burns or full-thickness injuries with autologous skin cell suspension (ASCS) achieves robust wound closure while limiting the amount of donor skin compared with standard autografting. A Next Generation Autologous Cell Harvesting Device (NG-ACHD) was developed to standardize the preparation process for ASCS to ensure biological attributes are obtained known to correlate with well-established safety and performance data. This study compared ASCS prepared using the NG-ACHD and ACHD following the manufacturer's guidance, evaluating cellular yields, viability, apoptotic activity, aggregates, phenotypes and functional capacity. Non-inferiority was established for all biological attributes tested and comparable healing trajectories were demonstrated using an in vitro skin regeneration model. In addition to standardization, the NG-ACHD also provides workflow efficiencies with the potential to decrease training requirements and increase the ease of incorporation and utilization of ASCS in clinical practice.

Telomerase mRNA Enhances Human Skin Engraftment for Wound Healing.

Deep skin wounds represent a serious condition and frequently require split-thickness skin grafts (STSG) to heal. The application of autologous human-skin-cell-suspension (hSCS) requires less donor skin than STSG without compromising the healing capacity. Impaired function and replicative ability of senescent cutaneous cells in the aging skin affects healing with autologous hSCS. Major determinants of senescence are telomere erosion and DNA damage. Human telomerase reverse transcriptase (hTERT) adds telomeric repeats to the DNA and can protect against DNA damage. Herein, hTERT mRNA lipid nanoparticles (LNP) are proposed and evaluated for enhancing cellular engraftment and proliferation of hSCS. Transfection with optimized hTERT mRNA LNP system enables delivery and expression of mRNA in vitro in keratinocytes, fibroblasts, and in hSCS prepared from donors' skin. Telomerase activity in hSCS is significantly increased. hTERT mRNA LNP enhance the generation of a partial-thickness human skin equivalent in the mouse model, increasing hSCS engraftment (Lamin) and proliferation (Ki67), while reducing cellular senescence (p21) and DNA damage (53BP1).

Development of microfabricated dermal epidermal regenerative matrices to evaluate the role of cellular microenvironments on epidermal morphogenesis.

Topographic features at the dermal-epidermal junction (DEJ) provide instructive cues critical for modulating keratinocyte functions and enhancing the overall architecture and organization of skin. This interdigitated interface conforms to a series of rete ridges and papillary projections on the dermis that provides three-dimensional (3D) cellular microenvironments as well as structural stability between the dermal and epidermal layers during mechanical loading. The dimensions of these cellular microenvironments exhibit regional differences on the surface of the body, and quantitative histological analyses have shown that localization of highly proliferative keratinocytes also varies, according to the regional geometries of these microenvironments. In this study, we combined photolithography, collagen processing, and biochemical conjugation techniques to create microfabricated dermal epidermal regeneration matrices (µDERMs) with features that mimic the native 3D cellular microenvironment at the DEJ. We used this model system to study the effect of the 3D cellular microenvironment on epithelialization and basal keratinocyte interaction with the microenvironment on the surface of the µDERMs. We found that features closely mimicking those in high-friction areas of the body (deep, narrow channels) epithelialized faster than features mimicking low-friction areas. Additionally, when evaluating ß1 expression, an integrin involved in epidermal morphogenesis, it was found that integrin-bright expression was localized in the depths of the features, suggesting that the µDERMs may play a role in defining cellular microenvironments as well as a protective environment for the regenerative population of keratinocytes. The outcomes of this study suggest that µDERMs can serve as a robust biomimetic model system to evaluate the roles of the 3D microenvironment on enhancing the regenerative capacity and structural stability of bioengineered skin substitutes.

Carbodiimide conjugation of fibronectin on collagen basal lamina analogs enhances cellular binding domains and epithelialization.

To improve the regenerative potential of biomaterials used as bioengineered scaffolds, it is necessary to strategically incorporate biologically active molecules that promote in vivo cellular processes that lead to a fully functional tissue. This work evaluates the effects of strategically binding fibronectin (FN) to collagen basal lamina analogs to enhance keratinocyte functions necessary for complete skin regeneration. We found that FN that was passively adsorbed to collagen-glycosaminoglycan basal lamina analogs enhanced epithelial thickness and keratinocyte proliferation compared with nontreated basal lamina analogs at 3 days of air/liquid (A/L) interface culture. Additionally, we evaluated the availability of FN cellular binding site domains when FN was either passively adsorbed or [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride] conjugated to basal lamina analogs fabricated from collagen-glycosaminoglycan coprecipitate or self-assembled type I collagen. It was found that 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride conjugation of FN significantly enhanced FN binding site presentation as well as epithelial thickness. Overall, the results gained from this study will be used to improve the regenerative capacity of basal lamina analogs for bioengineered skin substitutes as well as the development of bioengineered scaffolds for other tissue engineering applications.

Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment.

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.

Multiphoton excited fabricated nano and micro patterned extracellular matrix proteins direct cellular morphology.

We use multiphoton excited (MPE) photochemistry to fabricate patterned extracellular matrices (ECM) and to investigate the morphology of human dermal fibroblasts adhered to the resulting photocrosslinked linear structures of fibronectin (FN), fibrinogen (FG), and bovine serum albumin (BSA). These proteins were chosen to systematically investigate the roles of topography and ECM biochemistry on cell spreading, as fibroblasts bind directly to both FN and FG at RGD sites through known integrins, whereas BSA provides no comparable ECM cues for cell binding. MPE crosslinked patterns are created from parallel linear structures 600 nm in width, 200 microm in length, and spaced by either 10 or 40 microm. Immunofluorescence staining of FN and FG was used to assay the functionality of crosslinked proteins. The metrics of orientation, elongation, and cell perimeter were used to quantitate the resulting cellular behavior on the crosslinked protein patterns. These parameters all reflect statistical differences for cells on BSA, relative to the similar statistical behavior on fibronectin and fibrinogen. Cells on the BSA patterns are constrained by physical guidance and orientation between linear structures. In contrast, cells adhered on both FN and FG had a greater propensity to spread across adjacent structures, indicating the importance of cell matrix interactions. Focal adhesion staining of cells adhered to the protein structures revealed similar trends. These findings are consistent with our hypothesis that these crosslinked matrix protein structures are expected to direct cell adhesion and spreading and that the topography and ECM cues lead to different forms of guidance.

Multiphoton excited fabrication of collagen matrixes cross-linked by a modified benzophenone dimer: bioactivity and enzymatic degradation.

Multiphoton excited (MPE) photochemistry is used to fabricate model tissue engineering scaffolds directly from types I, II, and IV collagen. A modified benzophenone dimer (BPD) provides the photoactivation and becomes incorporated into the resulting collagen matrixes. Unlike xanthene photochemistries, the benzophenone dimer can be used in acidic environments, where most forms of collagen have the greatest solubility. The minimum feature sizes are investigated by using two- and three-photon excitation, where the latter provides for superior "resolution" and suggests that collagen structures can be fabricated on the size scales of focal contacts. The resulting structures display excellent retention of bioactivity as evidenced by highly specific cell adhesion as well as immunofluorescence labeling. Structural and chemical aspects of the collagen matrixes are probed through measuring the enzymatic degradation through specific and nonspecific proteases, as the resulting relative rates are consistent with the activity of these enzymes. The degradation rates can also be controlled through varying the cross-link density in the matrixes, which is achieved through tuning the exposure dose during the fabrication process. The degradation rates are also found to be consistent with swelling/shrinking measurements and thus the average mesh size of the matrixes. In all cases the enzymatic degradations are well-fit single exponentials, suggesting that the matrixes can be fabricated with a priori knowledge of their structural properties. These results coupled with the resulting bioactivity suggest that the multiphoton fabrication process may be a powerful tool for the creation of cell-sized tissue engineering scaffolds.